Although expression of the viral Vif protein is critical for HIV-1 replication in primary cells, the mechanism of action of Vif has, until recently, been largely unclear. However, recent data from the Malim laboratory suggest that Vif functions by blocking the ability of a host factor called CEM15 or APOBEC3G to compromise the infectivity of progeny virions. APOBEC3G is not expressed in certain cell lines that are permissive for Vif- HIV-1 replication and expression of APOBEC3G in trans is sufficient to render these cells non-permissive for Vif- HIV-1. Although the mechanism of action of APOBEC3G, or how Vif prevents this action, remains unknown, APOBEC3G belongs to a family of cytidine deaminases that includes a member known to edit mRNA. We therefore hypothesize that APOBEC3G may block HIV-1 replication by editing HIV-1 mRNA and inducing a mutational error catastrophe. Alternately, APOBEC3G may inhibit HIV-1 infectivity after being packaged into virions and directly interfering with an early step in the viral infection cycle. The focus of this grant is on determining how APOBEC3G blocks HIV-1 replication, how Vifprevents this block and finally to examine whether APOBEC3G contributes to HIV-1 species tropism. We have already demonstrated a specific interaction between HIV-1 Vif and hAPOBEC3G in vivo and will further characterize this interaction in vitro, including testing whether it is direct. We will test whether APOBEC3G is a cytidine deaminase and whether this activity is required for its effect on HIV-1 replication. We will also sequence HIV-1 genomic RNAs produced in the presence of APOBEC3G, and absence of Vif, to detect editing. We will test whether non-human, including simian, forms of APOBEC3G inhibit HIV-1 replication and are responsive to HIV-1 Vif. Based on evidence showing that murine APOBEC3G blocks Vif- HIV-1 infectivity yet is not responsive to Vif, we intend to construct chimeric enzymes derived from Vif-responsive human and Vif-non-responsive animal APOBEC3G variants to map residues critical for Vif function. We will also attempt to use genetic and biochemical techniques to identify the viral nucleic acid or protein sequences that cause HIV-1 to be specifically targeted by APOBEC3G. Together, this wide-ranging series of experiments should lead to considerable insight into how HIV-1 Vif functions and potentially suggest approaches to inhibit Vif function.